BIOL710: Molecular Biology Mondays and Wednesdays, 5:30PM LECTURERS SUGGESTED TEXT Mitchell Goldfarb HN834 772-5289 Goldfarb@genectr.hunter.cuny.edu Paul Feinstein HN904 650-3169 Feinstein@genectr.hunter.cuny.edu Biochemistry, 6th edition Berg, Tymockzo, & Stryer ISBN: 0716787245 OR Biochemistry, 4th edition Stryer ISBN: 0716720094 Available online from www.barnesandnoble.com www.amazon.com COURSE WEBSITE http://biology.hunter.cuny.edu/molecularbio/ Scope of the Course How are biological macromolecules synthesized and assembled? How do different macromolecules generate the structure of cells? How do proteins fold to acquire functionality? How do enzymes catalyze reactions? How is energy harvested and stored in the cell? How do pumps and channels store energy and control the chemical composition of cellular compartments? Intracellular biochemical signaling by proteins and lipids. DNA structure and replication. Transcription and post-transcriptional RNA modification. Regulation of transcription. The genetic code and protein translation. Similarities and differences in processing of genetic information in prokaryotes vs. eukaryotes LECTURE 1: pH, pK, Amino Acids, Peptide Properties, pI Reading: Berg Chapters 1,2 Before being able to discuss the properties of biological macromolecules, we must first review: 1) Covalent and noncovalent bonding 2) Oxidation and reduction 3) Acid/base chemistry Elemental Properties H Hydrogen Carbon Oxygen H C O Electropositive Forms 1 covalent bond Forms 4 covalent bonds H H O H C C H H H H O C C Sulphur N Strongly electronegative Forms 2 covalent bonds S Weakly electronegative Forms 3 covalent bonds Weakly electronegative Forms 2 covalent bonds P In oxidized form Forms 5 covalent bonds N H H H H R -- O -- P -- O -- H - H O C C phosphate side group O H glycine H O O H acetic acid N Phosphorus O H H Nitrogen ethanol H O C C C H S H O H cysteine Oxidation and Reduction of Organic Compounds and Oxygen OXIDATION -- Removal of an electron pair from a molecule REDUCTION -- Addition of an electron pair from a molecule In any oxidation/reduction reaction, one component (electron donor) gets oxidized, another component (electron acceptor) gets reduced H H O H C C H H H - 2e + 2H + H O C C H ETHANOL (reduced) H ACETALDEHYDE (oxidized) O O H O O2 H + ACCEPTOR 2NADH DONOR + 2H+ H 2H2O REDUCED + 2NAD+ OXIDIZED Weak Acids and Bases When hydrogen is covalently bonded to an electronegative atom (typically O or N) in a compound, the hydrogen proton may dissociate. In this situation: The protonated compound is called an ACID, and the deprotonated compound is called a BASE. Conjugate ACID Conjugate BASE H H H O C C H acetic acid O H H H O H H C C N H H H + hydroxyethylammonium H O C C H H H + H O - acetate + H O H H C C H H H N 2-amino-ethanol H pH Water is in equilibrium with its minor dissociation products, H+ and OHWhile concentration of H2O is 55 M, [H+][OH-] = 10-14 M In a neutral solution, [H+] = [OH-] = 10-7 M In an acidic solution, [H+] >> [OH-] . E.g. [H+] = 10-3 M , and [OH-] = 10-11 M) In a basic solution, [H+] << [OH-] . E.g. [H+] = 10-10 M , and [OH-] = 10-4 M) pH = - log10 [H+] At neutrality, pH = - log10 [10-7] = 7 Acidic solutions have pH < 7; Basic solutions have pH > 7 Generally speaking “weakly acidic” means pH = 4 to 5.5 “strongly acidic” means pH < 3 “weakly basic” means pH = 8.5 to 10 “strongly basic” means pH > 11 Strong Acids and Bases A strong ACID is an INORGANIC compound that FULLY DISSOCIATES into H+ and its negative counterion in water. H+ HCl Cl- H+ HNO3 HYDROCHLORIC ACID NO3- NITRIC ACID A strong BASE is an METAL HYDROXIDE compound that FULLY DISSOCIATES into OH- and its positive counterion in water. NaOH OH- Na+ 2OH- Ca(OH)2 SODIUM HYDROXIDE CALCIUM HYDROXIDE pH of strong acid and base solutions pH = - log10 [ACID] Ca+2 pH = 14 + log10 [BASE] e.g. e.g. pH of 10 mM HCl = 2 pH of 0.1 M NaOH = 13 TITRATION OF A STRONG ACID SOLUTION WITH STRONG BASE Different amounts of NaOH added to a 10mM HCl solution pH stays strongly acidic until nearly 10mM NaOH added, then swings steeply past neutral to strongly basic when NaOH exceeds 10mM NO CONTROL OF pH IN THE MILD ACID TO MILD BASE RANGE pH NEUTRAL 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 0 1 Molar Equivalents of NaOH 2 pKa of an acid/base conjugate pair H+ + HA Acid Ka = [H+ ][A- ] Proton TAKE LOGARHYTHM MULTIPLY BY -1 [HA] ABase [A-] pH = pKa + log10 [HA] IMPLICATIONS FOR ANY PARTICULAR ACID/BASE PAIR For a group with an acidic pKa, the base conjugate predominates at neutral pH For a group with an basic pKa, the acid conjugate predominates at neutral pH When acid and base are at equal concentrations, pH = pKa An acid/base pair acts as a buffer of strong acids or bases in pH range of pKa + 1. Titration of a Weak Acid Solution with a Strong Base The weak acid BUFFERs the effect of the strong base, keeping pH in the range of the pKa over wide range of base concentratiion As NaOH is added to a weak acid solution HA, the NaOH converts HA to A HA + NaOH - H2O + Na+ + A- Since pH = pKa + log [ A- ]/[ HA ] , the weak acid buffers in the range of pKa 7 For acetic acid pKa = 4.0 100 6 5 2 25 1 0 0 0.5 1 Molar Equivalents of NaOH pH - For unbuffered 0.1 M acetic acid pH = 2.5 50 3 [A ] (mM) 4 [HA] (mM) pKa 75 Three Types of Non-Covalent Bonding Influence Intramolecular and Intermolecular Interactions ELECTROSTATIC BONDING Oppositely charged groups are attractive. HYDROGEN BONDING A hydrogen covalently bonded to O or N can noncovalently interact with a O or N, if all three atoms are aligned and at appropriate distance. This is a hydrogen bond. H O R O H O R H H Three Types of Non-Covalent Bonding Influence Intramolecular and Intermolecular Interactions VAN DER WAAL’S FORCES AND HYDROPHOBIC INTERACTIONS A weak interaction between nonpolar molecular surfaces. Van der Waal’s forces contribute to favorability of hydrophobic interactions. The other crucial contributing factor is that interaction between two hydrophobic surfaces in a solution reduces the hydrophobic surface area and therby INCREASES the number of water-to-water solvent hydrogen bonds!!! AMINO ACIDS pK of carboxylic acid group is ~ 3.0 pK of amino group is ~ 9.5 POLYPEPTIDES FREE ROTATING All polypeptides synthesized from L-amino acids FREE ROTATING CLASSES OF AMINO ACIDS All proteins are synthesized from a pool of 20 amino acids (some additional amino acids are generated by modifications within synthesized polypeptides) Amino acids can be functionally grouped by properties of side chains (R) (a few amino acids fit overlap into more than one group) GROUPINGS ALIPHATIC -- Side chains participate in hydrophobic interactions AROMATIC -- Hydrophobic interactions and hydrogen bonding ACIDIC -- Ionic and hydrogen bonding BASIC -- Ionic and hydrogen bonding HYDROXYL -- Hydrogen bonding and sites of phosphate or sugar modifications AMIDO -- Hydrogen bonding and sites of sugar modifications SULPHUR -- Hydrogen bonding and sites of oxidative crosslinking “OTHER” -- Side chains confer specialized turning properties of polypeptide ALIPHATIC AMINO ACIDS NOTE: AROMATIC AMINO ACIDS Methionine is also a sulphurbearing amino acid NOTE: Tyrosine is also a hydroxylated amino acid HYDROXYLATED AMINO ACIDS Ser-, Thr-, or Tyr-OH in protein can undergo phosphate addition Ser- or Thr-OH in protein can can be site for carbohydrate addition (termed O-linked glycosylation) ACIDIC AMINO ACIDS Carboxylic acid side chain in Asp and Glu has pKa ~ 4.5 and carries a full -1 charge at neutral pH AMIDO AMINO ACIDS Asn is amidated version of Asp Gln is amidated version of Gln Asn and Gln are NOT charged, but are higly polar NH2 group on Gln in proteins can be site for carbohydrate addition (N-linked glycosylation) BASIC AMINO ACIDS Amino side chain of Lys and imino side chain of Arg have pK > 10 and carry a full +1 charge at neutral pH Ring imino side chain of His has pK ~ 6.5 And carries on average a fractional positive charge at neutral pH SULPHUR-CONTAINING AMINO ACIDS Methionine is also considered an aliphatic amino acid Nearby cysteines on the same or different polypeptide chains can undergo oxidation to generate a covalent DISULPHIDE bond - 2e + 2H + PROLINE IMPARTS INFLEXIBILITY ON REGION OF POLYPEPTIDE The propyl side chain of Proline is covalently bonded to the beta-nitrogen to form a ring. Technically, proline is an IMINO acid FREE ROTATING FREE ROTATING Beta-carbon at most amino acid residues have TWO rotatable bonds FREE ROTATING Beta-carbon at proline residues has only ONE rotatable bond AMINO ACID COMPOSITION AND SOLUTION pH DETERMINE POLYPEPTIDE CHARGE A protein’s ISOELECTRIC POINT (pI) is the pH at which the protein’s NET CHARGE = 0 An “acidic protein” has pI < 7 A “basic protein” has pI > 7 A protein has NET NEGATIVE CHARGE when pH > pI NET POSITIVE CHARGE when pH < pI pI is determined by a protein’s amino acid composition. Different pIs of different polypeptides can be used to separate these proteins by electrophoresis at specific pHs. Eg., an acidic protein has more acidic residues than basic ones NEXT LECTURE: PROTEIN STRUCTURE 1 Reading: Berg, Chapter 2